Myeloma-Specific Multiple Peptides Able to Generate Cytotoxic T Lymphocytes: A Potential Therapeutic Application in Multiple Myeloma and Other Plasma Cell Disorders

Multiple myeloma is a malignant disorder characterized by a multifocal proliferation and clonal expansion of long-lived plasma cells within the bone marrow, associated with skeletal destruction, serum monoclonal gammopathy, immunosuppression, and end-organ sequelae (1, 2). Despite aggressive chemotherapeutic regimens and novel therapies including immunomodulatory drugs (thalidomide, lenalidomide) and the proteasome inhibitor (bortezomib), development of acquired resistance to these agents is associated with refractory relapsed multiple myeloma (3). Alternatively, active-specific immunotherapy may provide more durable responses through induction of cytotoxic T lymphocytes (CTL) targeting cancer cells (4, 5). Although immunotherapy has shown a therapeutic benefit in cancer (6–8), this option is suboptimal in multiple myeloma and requires further improvement. The major challenges in developing a successful multiple myeloma–specific immunotherapy include heterogeneity of tumor-associated antigens (TAA) expression, frequent mutations of specific TAAs, tumor escape mechanisms, changes in immune cell function, and variability of the human T-cell repertoire. Thus, we hypothesized that use of immunogenic HLA-A2–specific epitopes from multiple TAAs may enhance induction of antigens-specific CTLs targeting malignant plasma cells and associated therapeutic efficacy in HLA-A2⁺ patients with multiple myeloma.

As target antigens, we selected XBP1 (X-box–binding protein 1), CD138 (syndecan-1), and CS1 (CD2 subset 1, CRACC, SLAMF7, CD319), which are associated with multiple myeloma pathogenesis and are highly expressed on the tumor cells. First, we propose XBP1 as an attractive therapeutic target antigen as XBP1 is a basic leucine zipper–containing transcription factor, which is required for the terminal differentiation of B lymphocytes to plasma cells and is uniformly expressed in cells and cell lines of all patients with multiple myeloma (9, 10). This antigen has been implicated in the proliferation of malignant plasma cells and is differentially expressed between normal plasma cells and plasma cells from patients' with monoclonal gammopathy of undetermined significance (MGUS) or multiple myeloma (11, 12). High amounts of immunoglobulin produced by plasma cells evoke estrogen receptor (ER) stress, which in turn activates IRE1-mediated XBP1 expression and subsequently mRNA splicing during plasma cell differentiation (10, 13, 14). As a consequence, the relative mRNA expression levels of spliced XBP1 compared with unspliced XBP1 are higher in multiple myeloma than in normal plasma cells (9), making XBP1 as a potential therapeutic target. The second potential target antigen we propose for the development of multiple myeloma–specific immunotherapy is CD138, a transmembrane heparan sulfate–bearing proteoglycan expressed by most multiple myeloma cells. CD138 is critical for the growth of tumor cells by mediating cell–cell adhesion, binding multiple myeloma cells to molecules such as collagen and fibronectin in the extracellular matrix, as well as binding to growth factors and cytokines (15, 16). In patients with multiple myeloma, shed syndecan-1 accumulates in the bone marrow, and soluble syndecan-1 facilitates multiple myeloma tumor progression, angiogenesis, and metastasis in vivo. Therefore, targeting CD138 on malignant plasma cells to prevent or reduce high levels of syndecan-1 in the serum, an indicator of poor prognosis in multiple myeloma (17–19), may have a direct clinical benefit. Finally, CS1 is a cell surface glycoprotein of the CD2 family, which is highly and uniformly expressed by malignant plasma cells and has restricted expression in normal tissues (20–23). CS1 localizes to the uropods of polarized multiple myeloma cells, where it mediates adhesion of multiple myeloma cells to bone marrow stroma and other human multiple myeloma cells (24). On the basis of the universal expression of these functional antigens on multiple myeloma cells, we hypothesized that development of an immunotherapeutic strategy targeting XBP1, CD138, as well as CS1 antigens could represent a novel treatment option for multiple myeloma.

In previous studies, we have identified immunogenic HLA-A2–specific peptides derived from each of these target antigens including heteroclitic XBP1-unspliced (US)_184-192 (YISPWILAV; ref. 25), heteroclitic XBP1-spliced (SP)_367-375 (YLFPQLISV; ref. 25), native CD138_260-268 (GLVGLIFAV; ref. 26), and native CS1_239-247 (SLFVLGLFL) peptides (27). These selected peptides were highly immunogenic in ex vivo studies inducing antigen-specific CTLs, which specifically responded against HLA-A2⁺ multiple myeloma cells. In the current studies, we provide evidence that a cocktail of 4 HLA-A2–specific peptides derived from XBP1-US, XBP1-SP, CD138, and CS1 induces multipeptide-specific CTLs (MP-CTL) enriched for effector CD8⁺ T cells with distinct functional immunogenic properties against HLA-A2⁺ multiple myeloma cells. The ability to induce CTLs against multiple target epitopes using a combination of these 4 immunogenic peptides provides the framework for their potential use in targeted immunotherapy to improve outcome in patients with plasma cell–related disorders.

The multiple myeloma cell lines, McCAR, MM1S, and U266, were obtained from American Type Culture Collection. The T2 cell line, a human B- and T-cell hybrid expressing HLA-A2 molecules, was provided by Dr. J. Molldrem (University of Texas MD Anderson Cancer Center, Houston, TX). All cell lines were cultured in RPMI-1640 medium (Gibco-Life Technologies) supplemented with 10% fetal calf serum (FCS; BioWhittaker), 100 IU/mL penicillin, and 100 μg/mL streptomycin (Gibco-Life Technologies).

Mouse anti-human CD3, CD4, CD8, CCR7, CD45RO, CD69, CD107a, IFN-γ, and HLA-A2 monoclonal antibodies (mAb) conjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE), PerCP, PerCP-Cy5.5, allophycocyanin (APC), Pacific Blue, APC-H7, or PE-Cy7 were purchased from Becton Dickinson (BD)/Pharmingen or BD/Biosciences. Recombinant human interleukin (IL)-2, IL-4, IFN-α, and TNF-α were purchased from R&D Systems, and granulocyte macrophage colony-stimulating factor (GM-CSF) was obtained from Immunex.

Heteroclitic XBP1-US_184–192 (YISPWILAV), heteroclitic XBP1-SP_367–375 (YLFPQLISV), native CD138_260–268 (GLVGLIFAV), and native CS1_239–247 (SLFVLGLFL) peptides were derived from XBP1-unspliced (US), XBP1-spliced (SP), CD138, and CS1 antigens, respectively. Influenza virus matrix protein_58–66 (GILGFVFTL) and CMV pp65 (NLVPMVATV) were selected as HLA-A2–specific control peptides. All peptides were synthesized by standard fmoc (9-fluorenylmethyl-oxycarbonyl) chemistry, purified to more than 90% using reverse-phase chromatography and validated by mass spectrometry for molecular weight (Biosynthesis). Lyophilized peptides were dissolved in dimethyl sulfoxide (DMSO; Sigma), diluted in AIM-V medium (Gibco-Life Technologies), and stored at −140°C.

A cocktail of 4 HLA-A2 peptides, including heteroclitic XBP1-US_184–192, heteroclitic XBP1-SP_367–375, CD138_260–268, and CS1_239–247, was evaluated for binding affinity using the T2 cell line, as described elsewhere (28). In brief, T2 cells were pulsed overnight with the multipeptide cocktail (0, 6.25, 12.5, 25, 50 μg/mL) plus 3 μg/mL human β2-microglobulin (Sigma). Following incubation, cells were stained with anti-human HLA-A2-FITC mAb and analyzed using a FACSCanto II flow cytometer (Becton Dickinson).

The multipeptide cocktail was examined for HLA-A2 stability, as described elsewhere (29). Briefly, T2 cells were pulsed overnight with the multipeptide cocktail (25 μg/mL) plus 3 μg/mL human β2-microglobulin, and the peptide/HLA-A2 complex stability was measured at 0, 2, 4, 6 and 14 hours after brefeldin A (BFA) treatment by staining cells with mouse anti-human HLA-A2-FITC mAb and flow cytometric analysis.

Monocyte-derived dendritic cells were generated as described elsewhere (30), with minor modifications. Briefly, monocytes isolated from peripheral blood mononuclear cells (PBMC) were cultured for 7 days in the presence of 1,000 U/mL GM-CSF and 1,000 U/mL IL-4 in RPMI-1640 medium (Gibco-Life Technologies) supplemented with 10% FCS. Fresh media plus GM-CSF and IL-4 was added to the cultures every other day. Mature dendritic cells (mDC) were obtained by adding 1,000 U/mL IFN-α plus 10 ng/mL TNF-α, along with fresh GM-CSF and IL-4 in 10% FCS-RPMI, on day 7 and then incubating for an additional 3 days.

CD3⁺ T cells were obtained from HLA-A2⁺ normal donors by negative selection using the EasySep magnet and RoboSep from StemCell Technologies. In brief, PBMCs were depleted of B cells, monocytes, natural killer (NK) cells, erythroid cells, platelets, and basophils using a cocktail of bispecific tetrameric antibody complexes. After the removal of magnetically labeled unwanted cells, the enriched CD3⁺ T cells were washed and examined by flow cytometry.

Primary CD138⁺ cells were isolated from bone marrow mononuclear cells obtained from both HLA-A2⁺ and HLA-A2⁻ patients with multiple myeloma using RoboSep CD138-positive immunomagnetic selection technology (StemCell Technologies), after appropriate informed consent.

MP-CTLs were generated ex vivo by repeated multipeptide stimulation of CD3⁺ T lymphocytes obtained from HLA-A2⁺ normal donors. In brief, antigen-presenting cells (APCs; mDC or T2 cells) pulsed overnight with a cocktail of heteroclitic XBP1-US_184–192, heteroclitic XBP1-SP_367–375, CD138_260–268, and CS1_239–247 peptides (25 μg/mL total; 6.25 μg/mL/peptide) were irradiated (20 Gy) and then used to prime autologous CD3⁺ T cells at a 1:20 APCs/MP-to-CD3⁺ T-cell ratio in AIM-V medium supplemented with 10% human AB serum. Cultures were restimulated every 7 days with irradiated APCs/MP for a total of 4 cycles to generate MP-CTLs. IL-2 (50 U/mL) was added to the cultures 2 days after the second stimulation and was replenished until the culture was completed.

One week after the fourth stimulation, MP-CTLs and control T cells were evaluated for total CD3⁺CD8⁺ T cells or naive, effector memory, and activated CD3⁺CD8⁺ T cells by staining with CD3-PacBlue, CD8-APC-H7, CCR7-PeCy7, CD45RO-PE, and/or CD69-PerCP mAbs. After staining, the cells were washed, fixed in 2% paraformaldehyde PBS, and analyzed by flow cytometry.

CD107a degranulation and IFN-γ–producing CD8⁺ CTLs were identified by cell surface marker and intracellular cytokine staining by flow cytometry. Briefly, MP-CTLs or control T cells were stimulated with HLA-A2⁺ McCAR or U266 multiple myeloma cell lines or with K562-A*0201 cells pulsed with respective peptide in the presence of CD107a mAb. After 1-hour incubation, CD28/CD49d mAb (BD), as well as protein transport inhibitors, BFA (BD) and monensin (BD), were added to the cultures and incubated for an additional 5 hours. As a baseline control, MP-CTLs were cultured in media with CD28/CD49d mAb, BFA, and monensin alone. After incubation, cells were stained with CD3-PacBlue and CD8-APC-H7, CCR7-PeCy7, CD45RO-PE, and/or CD69-PerCP anti-human mAbs, followed by fixation/permeabilization (Cytofix/Cytoperm, BD) and stained with anti-IFN-γ FITC mAb to detect intracellular cytokine production. Finally, cells were washed with Perm/Wash solution (BD), fixed in 2% paraformaldehyde, and analyzed by flow cytometry.

Proliferation of MP-CTLs was evaluated using carboxy fluorescein succinimidyl ester (CFSE), as described elsewhere (31) with minor modifications. In brief, MP-CTLs labeled with CFSE (Molecular Probes) were incubated with stimulator cells (from cells or cell lines of HLA-A2⁺ CD138⁺ patients with multiple myeloma). As a control, MP-CTLs labeled were cultured in media alone. After a 5-day incubation, cells were harvested and stained with anti-CD3 and anti-CD8 mAbs and evaluated by flow cytometry to measure proliferation.

The cytotoxic activity of MP-CTLs was measured using a calcein release cytotoxicity assay, as described previously (32) with minor modifications. Briefly, target cells were labeled with calcein-AM (Molecular Probes) and incubated for 4 hours with MP-CTLs at various effector:target cell ratios in 96-well, U-bottom microtiter plates (triplicate wells/sample). The fluorescence of each supernatant was monitored at 490 nm excitation and 520 nm emission wavelengths using a VICTOR²-1420 multilabel counter (PerkinElmer). Cytotoxicity of CTLs was calculated as follows: % specific lysis = [(experimental release − spontaneous release)/(maximum release − spontaneous release)].

Results are presented as mean ± SE. Groups were compared using unpaired Student t test. Differences were considered significant when P < 0.05.

The 4 immunogenic peptides including heteroclitic XBP1-US_184–192 (YISPWILAV), heteroclitic XBP1-SP_367–375 (YLFPQLISV), native CD138_260–268 (GLVGLIFAV), and native CS1_239–247 (SLFVLGLFL; Table 1) have been individually shown to induce an immune response. Here, we have evaluated the peptides as a multipeptide cocktail. The HLA-A2–specific binding and stability of the multipeptide cocktail was evaluated by measuring upregulation of HLA-A2 molecules on T2 cells by flow cytometry and compared with affinity of the HLA-A2–specific control influenza virus matrix protein (IVMP)_58–66 peptide. The peptide-binding assay showed an increase in HLA-A2 mean fluorescence intensity (MFI) on T2 cells in a dose-dependent manner (0–50 μg/mL), reaching a plateau at a total peptide concentration of 25 μg/mL (6.25 μg/mL/peptide; MFI: 10,787.33 ± 2,371.71), which was similar to the highest total peptide concentration tested, 50 μg/mL (MFI: 10,889.33 ± 2,888.48; Fig. 1A). Therefore, a total multipeptide concentration of 25 μg/mL (6.25 μg/mL/peptide) was selected for evaluation of HLA-A2–binding stability.

In the peptide-binding stability assay, T2 cells were pulsed overnight with 25 μg/mL of the multipeptide cocktail, washed to remove unbound peptides, and then treated with BFA to block cell surface expression of newly synthesized HLA-A2 molecules. T2 cells were then evaluated for their HLA-A2 MFI at 0, 2, 4, 6, or 14 hours post-BFA treatments. Flow cytometric analysis show that stability of the multipeptide cocktail was highly maintained up to 6 hours post-BFA treatment (MFI: 0 hour = 9,726.00 ± 1,373.24, 2 hours = 9,132.33 ± 1,435.51, 4 hours = 9,125.33 ± 1,130.62, 6 hours = 8,818.67 ± 413.50; Fig. 1B). At 14 hours post-BFA treatment, HLA-A2–specific affinity of multipeptide cocktail was decreased but was still greater (MFI: 6,793.67 ± 1,617.01) than affinity of the control IVMP_58–66 peptide (MFI: 4,921.33 ± 1,428.16). On the basis of these results, we confirmed a high level of HLA-A2–specific affinity and stability of the multipeptide cocktail and then proceeded to further evaluate the cocktail for its immunogenicity and ability to induce multiple myeloma–specific CTLs.

Flow cytometric analyses showed that MP-CTLs contained a higher proportion of CD3⁺CD8⁺ T cells (donor 1: 86%, donor 2: 74%) compared with control T-cell cultures (donor 1: 25%, donor 2: 25%; Fig. 2). We also observed distinct phenotypic changes in the CD3⁺CD8⁺ T-cell subset within the MP-CTLs. The frequency of effector memory T cells (EM: CD45RO⁺CCR7⁻/CD3⁺CD8⁺) was increased (donor 1: control 5% vs. MP-CTL 44%, donor 2: control 4% vs. MP-CTL 35%), associated with a corresponding decrease in naive T cells (CD45RO⁻CCR7⁺/CD3⁺CD8⁺; donor 1: control 74% vs. MP-CTL 8%, donor 2: control 60% vs. MP-CTL 6%). In addition, we observed an increase in the frequency of activated CD69⁺/CD3⁺CD8⁺ T cells within the MP-CTLs as compared with the control T-cell cultures (donor 1: control 3% vs. MP-CTL 39%, donor 2: control 5% vs. MP-CTL 13%; Fig. 2). Thus, these results show that repeated stimulation of CD3⁺ T cells with the multipeptide cocktail composed of XBP1-US, XBP1-SP, CD138, and CS1-specific peptides results in distinct phenotypic changes and expansion of CD3⁺/CD8⁺ T-cell subsets characteristic of antigen-specific CTLs.

In previous studies, we have showed that HLA-A2–restricted and multiple myeloma–specific IFN-γ production by CTLs generated specifically with XBP1-US, XBP1-SP, CD138 or CS1 peptide in response to cells or cell lines of patients with multiple myeloma (25–26, 27). In the current study, MP-CTLs were analyzed by flow cytometry for their ability to produce intracellular IFN-γ upon stimulation with the HLA-A2⁺ multiple myeloma cell lines. Both EM (CD45RO⁺CCR7⁻) and activated (CD69⁺) CD3⁺CD8⁺ T cells within the MP-CTLs produced IFN-γ in response to HLA-A2⁺ multiple myeloma cell lines (Fig. 3). The frequency of IFN-γ–producing cells was increased upon stimulation with either McCAR cells [donor 1: control vs. MP-CTL: 0% vs. 4.7% EM cells, 0.8% vs. 6.1% activated cells; donor 2: 0.2% vs. 2.7% EM cells, 1% vs. 3.9% activated cells] or U266 cells [donor 1: control vs. MP-CTL: 0% vs. 8% EM cells, 0% vs. 11.2% activated cells; donor 2: 0.4% vs. 2.9% EM cells, 1.3% vs. 3.0% activated cells]. The naive (CD45RO⁻CCR7⁺) CD3⁺CD8⁺ T cells within the MP-CTLs showed a minimum level of IFN-γ production when stimulated with the HLA-A2⁺ multiple myeloma cell lines (data not shown).

The function of the MP-CTLs was analyzed using a CFSE proliferation assay. MP-CTL proliferation was measured on day 5, evidenced by a decrease in fluorescence of the CFSE-labeled MP-CTLs (Q1-gated cells) following stimulation with HLA-A2⁺ multiple myeloma primary cells or cell lines (Fig. 4). The MP-CTLs proliferated in response to CD138⁺ MM cells from 3 HLA-A2⁺ patients with multiple myeloma (proliferating cells: 33%, 29%, or 41%). In addition, MP-CTLs also proliferated in response to McCAR (proliferating cells: 57%) and U266 (proliferating cells: 49%) HLA-A2⁺ multiple myeloma cell lines. MP-CTLs cultured in media alone displayed only a low-level (5%) proliferation. Taken together, these data show the proliferation of MP-CTLs in stimulation with either cells or cell lines of HLA-A2⁺ patients with multiple myeloma.

Next, we evaluated the cytotoxic activity of MP-CTLs using a 4-hour calcein release assay. MP-CTLs generated from different HLA-A2⁺ donors' CD3⁺ T cells were evaluated for their cytotoxic activity against cells or cell lines of HLA-A2⁺ patients with multiple myeloma (Fig. 5). The cells of HLA-A2⁺ patients with multiple myeloma were effectively lysed by MP-CTLs at various effector:target cell ratios (donor A MP-CTL: patient #1: 0%–48%, patient #2: 9%–42%; donor B MP-CTL: patient #1: 0%–45%, patient #2: 1%–35%). In addition, MP-CTLs similarly show a high level of cytotoxic activity against McCAR cells (donor A MP-CTL: 8%–36%; donor B MP-CTL: 0%–74%:) and U266 (donor A MP-CTL: 0%–83%, donor B MP-CTL: 2%–43%) multiple myeloma cell lines. Compared with MP-CTLs, control CD3⁺ T cells from the same donors showed a significantly lower level of cytotoxicity against cells or cell lines of HLA-A2⁺ patients with multiple myeloma. In addition, MP-CTLs did not lyse MHC-mismatched tumor cells, including the HLA-A2⁻ multiple myeloma cell line (MM1S) or HLA-A2⁻ multiple myeloma cells from 3 different patients (data not shown). Taken together, these data confirm the HLA-A2–restricted cytotoxic activity of MP-CTLs against HLA-A2⁺ multiple myeloma cells.

Finally, we evaluated whether CTLs generated using multipeptide cocktail respond to each of the peptides individually. MP-CTLs were analyzed for their ability to degranulate (CD107a expression) and produce intracellular IFN-γ in response to heteroclitic XBP1-US_184–192 (YISPWILAV), heteroclitic XBP1-SP_367–375 (YLFPQLISV), native CD138_260–268 (GLVGLIFAV), and native CS1_239–247 (SLFVLGLFL) peptides. The analyses were conducted by measuring the specific MP-CTL response to K562-A*0201 cells (33) pulsed with the respective peptide. As controls, we used non–peptide-pulsed K562-A*0201 cells or K562-A*0201 cells pulsed with an irrelevant HLA-A2–specific CMV pp65 (NLVPMVATV) peptide. Figure 6A shows a representative flow cytometric analysis of the peptide-specific response from donor A MP-CTLs. The MP-CTLs showed a high proportion of CD107a⁺IFNγ⁺/CD3⁺CD8⁺ T cells (gated Q2) in response to XBP1-US (2.7%), CD138 (1.7%), and CS1 (12.5%) peptides but not to XBP1-SP (0.2%) peptide. No response was observed to the irrelevant CMV pp65 peptide (0.2%) or to the nonpeptide (0.2%) controls. Further analyses were conducted using MP-CTLs generated from 3 additional HLA-A2⁺ donors (Donors B, C, and D) for their CD107a degranulation or IFN-γ production in response to K562-A*0201 cells presenting each individual peptide (Fig. 6B). Specific responses were detected in MP-CTLs generated from each of these donors against all the relevant peptides but not to irrelevant HLA-A2–specific CMV pp65 peptide. However, variations were detected among the CTLs generated from different individuals in the level of specific response in degranulation and IFN-γ production to each relevant peptide. Therefore, these studies indicate that the multipeptide cocktail including XBP1-US, XBP1-SP, CD138, and CS1 epitopes can induce response to respective peptides, with specific CTLs targeting multiple antigens on HLA-A2⁺ multiple myeloma cells.

The discovery and application of novel immunogenic peptides offers a potentially new immunotherapeutic option, either as a vaccine or cellular therapy. The XBP1, CD138, and CS1 antigens have been implicated in multiple myeloma pathogenesis, and all are more highly expressed on cells of patients with multiple myeloma than on normal plasma cells. Indeed, the therapeutic potential of targeting these antigens has been evaluated with promising preclinical and clinical studies (34, 35). However, immunologic tolerance to antigens as self-proteins may inhibit development of an effective immune response and therefore be detrimental to developing an effective therapeutic strategy (36–38). To bypass tolerance and enhance peptide immunogenicity, we designed heteroclitic peptides YISPWILAV or YLFPQLISV from nonspliced or spliced XBP1 protein, respectively, which have higher HLA-A2 affinities than their originally identified native XBP1-US_184–192 (NISPWILAV) or XBP1-SP_367–375 (ELFPQLISV) peptides (25). In previous studies, we and others have shown that heteroclitic peptides can generate functional CTLs against tumor cells with cross-reactivity to their corresponding native peptides, suggesting their clinical applicability (29, 39, 40). In addition, the native peptides from CD138 and CS1 antigens, CD138_260–268 (GLVGLIFAV) and CS1_239–247 (SLFVLGLFL), were highly specific to the HLA-A2 locus, maintained a strong MHC/peptide stability complex, and induced functional anti–multiple myeloma CTLs (26, 27). Therefore, the CD138_260–268 and CS1_239–247 peptides were used in their native form and were not further modified. These peptides, especially in combination, may be useful for the development of a vaccine strategy to treat multiple myeloma–related diseases.

In the current studies, we evaluated the ability of a cocktail of these 4 HLA-A2 peptides to induce specific CTL responses against the respective multiple myeloma target antigens. We hypothesized that a multi-epitope vaccine would allow for a wider repertoire of tumor-associated peptides to be presented, thereby inducing a more robust immune response against tumor cells than vaccines specific to a single antigen, which may lose activity following specific antigen mutation or deletion on tumor cells. Moreover, this approach can also overcome the variation or absence of the appropriate T-cell repertoire, which can result in the lack of peptide-specific CTL induction to a single antigen-based vaccine. Therefore, we propose that a using a cocktail of immunogenic peptides capable of generating CTLs to multiple myeloma–associated antigens represents a more promising immunotherapeutic strategy.

We recognize the potential concern of epitope dominance and competition among these peptides specific to the same HLA molecules, which may impair or block the full spectrum of immune response against all of the target antigens (41). When using a mixture of peptides specific to the same MHC locus, a specific concern arises whether the lower affinity peptides will effectively bind and present in MHC molecules to induce T-cell responses in the presence of higher affinity peptides. In clinical trials of multipeptide vaccines, with each peptide has been administered at a different injection site to avoid this possibility of competition among peptides (42–44). However, this requirement might limit feasibility of this approach, as many novel peptides specific to TAAs have been defined. Here, we investigated whether the 4 peptides selected can be applied in combination to induce MP-CTLs. In these studies, we generated MP-CTLs ex vivo by stimulating HLA-A2⁺ normal donors' CD3⁺ T cells with APCs pulsed with a cocktail of the 4 immunogenic peptides. To avoid potential competition in HLA-A2 affinity among the specific peptides, we avoided excess concentrations of an individual peptide by using a minimum concentration of multipeptide (25 μg/mL total; 6.25 μg/peptide) to pulse the APCs during CTL generation. Our data showed that simultaneous pulsing with a 4-peptide cocktail did not compromise the functional immune activity of resultant MP-CTLs. Importantly, MP-CTLs showed specific functional activities, IFN-γ production, CD107 degranulation, and cell proliferation triggered by each relevant peptide but not to an irrelevant HLA-A2–specific CMV pp65 (NLVPMVATV) peptide. The XBP1-SP_367–375 and CS1_239–247 peptides, which have relatively lower HLA-A2 affinity, induced CTLs whose immunologic function was comparable with CTLs generated with the higher HLA-A2 affinity XBP1-US_184–192 and CD138_260–268 contained within the cocktail. Thus, we show that the possible competition among multipeptide, which bind to the same MHC class I molecules, does not compromise immunogenicity of lower affinity peptides.

These data suggest that immunogenic peptides administered in a mixture may generate functional CTLs in patients. In addition, several reports using a mixture of peptides with different HLA-A2 affinities further support our observation. For example, a prior study showed that CTLs generated by ex vivo stimulation with a peptide mixture show reactivity to 3 different peptides at a level comparable with that obtained by stimulation with each individual peptide separately (45). This study also showed that CTL recognition of lower affinity peptides specific to HLA-A2 molecules was maintained when target cells were copulsed with higher affinity peptides. Other investigators have also reported that competition among peptides for MHC binding does not significantly inhibit T-cell induction or activities (46, 47). Our in vitro studies used normal donor T cells and dendritic cells to optimize vaccine development. Importantly, defects in both T cells and dendritic cells have been described in patients with multiple myeloma. Thus, we are currently in the evaluation of the multipeptide for their ability to elicit tumor-specific immune response using patients' T cells and dendritic cells. In addition, their functional capacity to respond to multipeptide vaccine, as well as its clinical relevance, will be assessed in a clinical trial.

Other considerations that influence the success of vaccine trials include selection of an optimal adjuvant, inclusion of MHC class II–specific peptides, selection of the appropriate patient population, as well as concomitant chemotherapy, mAb therapy, or immunomodulatory drug therapy. For example, a previous phase III randomized trial did not show a superior clinical response to the gp100 peptide vaccine combined with ipilimumab (anti-CTLA4) compared with ipilimumab alone (48). However, the gp100 peptide vaccine showed an improved clinical response in a more recent phase III trial reported, when it was co-administered with IL-2 (8). This difference both highlights the need for further validation of these results trials and suggests important differences in adjuvant therapies administered with the vaccine. Besides anti-CTLA4 and IL-2, the efficacy of other adjuvants such as CpG, GM-CSF, IFN-α, and montanide ISA51 has been reported in various studies (7, 49–51). Importantly, combination studies with vaccines must be designed very carefully, as patients may already have received chemotherapy with long-lasting negative effects on their immune systems, thus weakening the potential benefit of a therapeutic vaccine. In our separate studies, we have shown that conventional chemotherapy used in multiple myeloma is detrimental to the function of immune-mediated responses (52, 53). However, our and other recent studies have shown that the immunomodulatory agent lenalidomide increases immune stimulatory properties and inhibits regulatory T cells in multiple myeloma (54); thus, the efficacy of a vaccine may be enhanced when used in combination with lenalidomide. In addition, induction of CD4⁺ T cell response using MHC class II peptides may be critical for establishing more long-term immunity to the HLA-A2–specific peptides (55, 56).

In summary, we have developed an immunotherapy targeting multiple TAAs using a cocktail comprised of XBP1-US, XBP1-SP, CD138, and CS1-specific epitopes, which may be applied in multiple myeloma and other plasma cell disorders. This proposed novel vaccine-based therapy will first be evaluated as an individual immunotherapy but may require additional incorporation of optimal adjuvants, MHC class II peptides, and/or immunomodulatory agents in suitable patient populations.

J. Bae, K.C. Anderson, and N.C. Munshi have ownership interest (including patents) and are consultant/advisory board members in OncoPep Inc. No potential conflicts of interest were disclosed by the other authors.

Conception and design: J. Bae, K.C. Anderson, N.C. Munshi

Development of methodology: J. Bae, J. Daley, K.C. Anderson, N.C. Munshi

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): J. Bae, J. Daley, Y-.T. Tai, K.C. Anderson, N.C. Munshi

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): J. Bae, J. Daley, Y-.T. Tai, K.C. Anderson, N.C. Munshi

Writing, review, and/or revision of the manuscript: J. Bae, K.C. Anderson, N.C. Munshi

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): J. Bae, R. Smith, N. Mimura, N.C. Munshi

Study supervision: J. Bae, K.C. Anderson, N.C. Munshi

This work was supported in part by grants from the NIH RO1-124929 to N.C. Munshi; P50-100007, PO1-78378, and PO1155258 to K.C. Anderson and N.C. Munshi; and RO1-50947 to K.C. Anderson. This research was also supported, in part, by a kind donation from Mr. and Mrs. Stewart Nagler. K.C. Anderson is an American Cancer Society Clinical Research Professor.